Because TNF a-stimulated gene-6 (TSG-6) modulates CD44-mediated cellular interactions with hyaluronan, we examined the possibility that TSP1 interacts with TSG-6. We found that recombinant full length human TSG-6 (TSG-6Q) and Link module of TSG-6 (Link_TSG6) bind 125I-TSP1 with comparable affinities. Trimeric recombinant constructs containing the N-modules of TSP1 or TSP2 inhibit binding of TSP1 to TSG-6Q and Link_TSG6, but other recombinant regions of TSP1 do not. Therefore, the N-modules of both TSP1 and TSP2 specifically recognize the Link module of TSG-6. Heparin, which binds to these domains of both proteins, strongly inhibits binding of TSP1 to Link_TSG6 and TSG-6Q, but hyaluronan does not. Inhibition by heparin is due to its binding to TSP1, because heparin also inhibits TSP1 binding to Link_TSG6 mutants deficient in heparin binding. Removal of bound Ca2+ from TSP1 reduces its binding to full-length TSG-6. Binding of TSP1 to Link_TSG6, however, is enhanced by chelating divalent cations. In contrast, divalent cations do not influence binding of the N-terminal region of TSP1 to TSG-6Q. This implies that divalent cation-dependence is due to conformational effects of Ca-binding to the C-terminal domains of TSP1. TSP1 enhances covalent modification of inter-alpha-trypsin inhibitor by TSG-6 and transfer of its heavy chains to hyaluronan, suggesting a physiological function of TSP1 binding to TSG-6 in regulation of hyaluronan metabolism at sites of inflammation. We identified a specific interaction between TSP1 and versican, that is induced during a toll-like receptor-3-dependent inflammatory response in vascular smooth muscle cells. TSP1 binding to versican is modulated by divalent cations. This interaction is mediated by interaction of the G1 domain of versican with the N-module of TSP1 but only weakly with the corresponding N-terminal region of TSP2. The G1 domain of versican contains two Link modules, which are known to mediate TNF a-stimulated gene-6 protein binding to TSP1, and the related G1 domain of aggrecan is also recognized by TSP1. Therefore, TSP1 interacts with three members of the Link-containing hyaladherin family. On the surface of poly-I:C-stimulated vascular smooth muscle cells, versican organizes into fibrillar structures that contain elastin but are largely distinct from those formed by hyaluronan. Endogenous and exogenously added TSP1 incorporates into these structures. Binding of exogenous TSP1 to these structures, to purified versican, and to its G1 domain is potently inhibited by heparin. At higher concentrations, exogenous TSP1 delays the poly-I:C induced formation of structures containing versican and elastin, suggesting that TSP1 negatively modulates this component of a vascular smooth muscle inflammatory response.